PROJECT SUMMARY Bacterial multidrug efflux transporters confer resistance to structurally diverse antimicrobials, which is one of the major causes for clinical treatment failure. Campylobacter jejuni is a major enteric pathogen and has developed various mechanisms for antibiotic resistance. Recently, both the World Health Organization and the Centers for Disease Prevention and Control have designated Campylobacter as a ?serious antibiotic resistance threat?. In Campylobacter, the multidrug efflux pump CmeABC, a RND-type efflux system, plays a key role in the resistance to various antimicrobials and in intestinal colonization by mediating bile resistance. In CmeABC, the three proteins assemble to form a powerful tripartite machinery, allowing direct efflux of substrates across both membranes of the Gram-negative cellular envelope. CmeABC is essential for C. jejuni as it has an important natural function for bile resistance, which is required for Campylobacter colonization in animal intestine. Typically, CmeABC requires to function cooperatively with other resistance mechanisms (such as target mutations) to confer clinically relevant antibiotic resistance. However, a ?super? resistance-enhancing variant of CmeABC (RE- CmeABC) has recently emerged in clinical isolates of C. jejuni. This variant pump has a distinct CmeB sequence and is much more potent in conferring multidrug resistance. Additionally, we found that RE-CmeABC is increasingly prevalent in clinical isolates and mediates exceedingly high-level resistance to fluoroquinolone, a clinically important antibiotic for treating campylobacteriosis. Our preliminary data further suggest the enhanced efflux function of RE-CmeABC is due to sequence variations in the RE-CmeB transporter. To begin to understand how CmeABC extrudes antimicrobials, we have initiated work to decipher the structural basis of CmeABC- mediated efflux. Our preliminary crystallization data indicate that CmeB forms a homotrimer, where individual protomers bind to and export substrates independently. Based on the solid preliminary data, we propose in this application to pursue three specific aims to 1) identify the specific mutations responsible for the enhanced efflux function in RE-CmeABC, 2) define the structural basis of CmeB-mediated antibiotic efflux and how sequence polymorphisms affect the structure-function relationship, and 3) determine the horizontal spread of RE-cmeABC and its impact on C. jejuni fitness in the absence and presence of antibiotic selection pressure. We will use a high throughput mapping strategy, a CRISPR-Cas9 based technique for efficient gene editing and replacement, in vitro and animal model systems, x-ray crystallography, and single-molecule FRET to achieve the goals of the three specific aims. The team of investigators have strong and complementary expertise, and are uniquely positioned to conduct the proposed work, which is expected to reveal novel mechanisms used by an RND-type transporter for antibiotic extrusion and enhanced multidrug resistance. This gained knowledge should be transferrable to other bacterial efflux pumps, and the findings may facilitate the development of new strategies to control the emergence and spread of multidrug resistant Campylobacter.